The main goal of this grant is to determine the mechanism of activation of the cell surface receptor Notch by transmembrane ligands of the Delta/Serrate/Lag-2 (DSL) superfamily. DSL-Notch signaling is an important and pervasive mechanism of intercellular communication, conserved in all multi-cellular animals, from sponges to man. It has enormous implications for human health, as genetic and environmental perturbations of DSL-Notch signaling cause a wide range of cancers, as well as developmental, immune, and neurological disorders. Thus, determining how DSL ligands activate Notch is critical for developing diagnostic and therapeutic tools to treat human disease, a central mission of the NIH. Our past work was instrumental in defining the basic mechanism of signal transduction by Notch. Using Drosophila as a model system, we discovered that Notch is a membrane tethered transcription factor that is cleaved in response to ligand, allowing the intracellular domain to enter the nucleus and turn on target genes. In the proposed work, we will address the still unanswered and crucial question of how ligand binding induces the initial cleavage responsible for activating the receptor. We will build on our previous discovery that to activate Notch on signal-receiving cells, DSL ligands must undergo endocytosis in signal-sending cells specifically by the adaptor protein Epsin. This finding, together with recent structural and biophysical studies, has suggested that ligand endocytosis by Epsin induces an allosteric change in the Notch codomain that exposes an otherwise buried cleavage site to the activating protease. In the proposed research we will use new approaches to manipulate ligand and receptor structure in vivo to test three hypotheses. First, that mechanical force generated across the intercellular ligand/receptor bridge is responsible for the allosteric change that renders the receptor susceptible to cleavage. Second, that this force is exerted by the ligand as it undergoes Epsin-dependent endocytosis into the sending cell. Third, that receptor plays an active role in generating this force by (i) inducing the ligand to enter the Epsin pathway, and (ii exerting an opposing force that depends on its own endocytosis. The results of these experiments will either establish the mechanical force model and the role of Epsin- dependent ligand endocytosis in generating the required force, or lead to other testable hypotheses for the basic mechanism by which ligand activates Notch. The innovative methods and reagents we generate will also be applicable to other problems in signal transduction and animal development.2